KR102039059B1 - Protein comprising ectodomain of japanese encephalitis virus envelope proteins, homodimer thereof, and use thereof - Google Patents

Abstract

The present invention relates to a protein comprising the protruding domain of the Japanese encephalitis virus coat protein, a homodimer thereof, and a pharmaceutical composition for preventing or treating a disease caused by the Japanese encephalitis virus infection comprising the same as an active ingredient. The present invention utilizes an insect cell expression system to provide homodimeric protein antigens of structurally stable overhang domains as originally present on the surface of a virus. In addition, the antigen may be used as a vaccine more stable than the conventional Japanese encephalitis vaccine because of its excellent ability to produce antibodies in the subject when injected into the subject, and may be used for diagnosis of Japanese encephalitis.

Description

Protein comprising the protruding domain of Japanese encephalitis virus coat protein, homodimer thereof, and use thereof {PROTEIN COMPRISING ECTODOMAIN OF JAPANESE ENCEPHALITIS VIRUS ENVELOPE PROTEINS, HOMODIMER THEREOF, AND USE THEREOF}

The present invention relates to a protein comprising a protruding domain of a Japanese encephalitis virus coat protein, a homodimer thereof, and a use thereof.

Japanese encephalitis virus (japanese encephalitis virus, JEV) is a Flaviviridae degwa (Flaviviridae), flaviviruses in positive virus of twenty-sided with a diameter of a virus, 40 nm to 50 nm that belongs to (Flavivirus). The viral surface consists of a membrane consisting of membrane protein (M), envelope protein (E), and lipids. The viral gene has a positive single stranded RNA and is about 11 kb in size.

In general, JEV causes symptoms of encephalitis, aseptic meningtis, and febrile headache. The symptoms increase with age and cause a high rate of encephalitis in elderly people over 60 years old. JEV is widespread throughout Asia, including Korea, Japan, China, Taiwan, Southeast Asia, India, and Russia, and has recently been reported in the West. However, no clear treatment is known to date.

The Japanese encephalitis virus vaccine has a total of three vaccines, such as two types of inactivated vaccines and live attenuated vaccines, such as vaccines derived from rat brain tissue and vaccines derived from hamster kidney cells. On the other hand, in South Korea, inactivated rat brain tissue-derived vaccines and attenuated vaccines using Nakayama strains have been used (Min Soo Park, et al., 1999). Currently, several new Japanese encephalitis vaccines are being developed. Representative vaccines are genetically modified vaccines and inactivated vaccines derived from Vero cells. Inoculation of inactivated vaccinia is followed by a different booster regime in each country after three base doses. Because inactivated vaccines are derived from mice, they can rarely cause shock to inoculators, and should be re-inoculated every two years because immunity is not maintained for a long time.

On the other hand, the purpose of diagnosing Japanese encephalitis can be divided into diagnosis of infection and surveillance of occurrence. For diagnosing Japanese encephalitis virus infection in a patient, virus isolation from a patient sample, measurement of antibody in blood, and gene detection methods can be performed. In order to monitor the occurrence of Japanese encephalitis, measurement of Japanese encephalitis-specific antibodies in pigs as amplification hosts and virus isolation tests in mosquitoes as a mediator are performed. Among antibody tests for viruses, neutralizing antibody titers are difficult to use as a general diagnostic method because they take a long time to determine. In addition, hemagglutination inhibition test method has the advantage of being able to quickly determine whether the Japanese encephalitis infection, but there are disadvantages that the preparation of the reagent is complicated and the results vary greatly depending on the tester. Therefore, there is a need for improvement of a test method for diagnosing Japanese encephalitis virus infection.

 Min Soo Park, et al., 1999. Immune Response to SA14-14-2 Live Attenuated Japanese Encephalitis Vaccine. Department of Pediatrics, Yonsei University College of Medicine. pp. 351-358.

Thus, the present inventors expressed and purified the insect cells in the form of a structurally stabilized coat protein, which is an antigenic protein of the Japanese encephalitis virus, for the serological diagnosis according to the Japanese encephalitis virus infection. The ELISA method was used to measure antibody titer with high sensitivity in serum of infected animals. In addition, the antibody produced after immunizing the purified recombinant coat protein to the experimental animal confirmed the neutralizing ability against the Japanese encephalitis virus, the present invention was completed.

It is an object of the present invention to provide homodimeric protein antigens which structurally stabilize the protruding domains of Japanese encephalitis virus coat proteins.

Another object of the present invention is to provide a pharmaceutical composition for diagnosing, preventing or treating Japanese encephalitis comprising an antibody against the antigen.

In order to achieve the above object, the present invention provides a homodimer protein in which two Japanese encephalitis virus coat protein overhang domains are combined.

The present invention also provides a vaccine for preventing or treating diseases caused by Japanese encephalitis virus infection comprising the homodimer protein as an active ingredient.

In addition, an antibody or fragment thereof that specifically binds to the homodimeric protein is provided.

Also provided is a kit for diagnosing Japanese encephalitis virus infection comprising the homodimer protein.

The present invention also provides a kit for diagnosing a Japanese encephalitis virus infection comprising an antibody that specifically binds to the homodimer protein.

Also provided is an expression vector comprising the nucleotides encoding the Japanese encephalitis virus coat protein protruding domain monomeric protein.

Also provided are host cells transfected with the expression vector.

The present invention also provides a method for producing a Japanese encephalitis virus coat protein protruding domain monomer protein comprising the step of obtaining a Japanese encephalitis virus coat protein protruding domain monomer protein from the transfected host cell.

In addition, (1) inducing an immune response by injecting the homodimer protein as an antigen into an experimental animal; And (2) provides a Japanese encephalitis virus antibody production method comprising the step of recovering the antibody from the experimental animal.

The present invention utilizes an insect cell expression system to provide homodimeric protein antigens of structurally stable overhang domains as originally present on the surface of a virus. In addition, the antigen may be used as a vaccine more stable than the conventional Japanese encephalitis vaccine because of its excellent ability to produce antibodies in the subject when injected into the subject, and may be used for diagnosis of Japanese encephalitis.

Figure 1 shows the comparative analysis of the coat protein amino acid sequence of Flavi virus using a protein sequencing comparison program (Clustal W & ESPript 3.0).
Figure 2 shows the comparative analysis of the coat protein amino acid sequence of the Japanese encephalitis virus using a protein sequencing comparison program (Clustal W & ESPript 3.0). At this time, the Japanese encephalitis virus coat protein protruding domain is 1 to 401.
Figure 3 shows the expression of the Japanese encephalitis virus coat protein overhang domain using Western blot.
FIG. 4A shows the purity of the Japanese encephalitis virus coat protein overhang domain after purification using Ni-NTA affinity chromatography.
Figure 4b is after purification of the Japanese encephalitis virus coat protein overhang domain using the Ni-NTA affinity chromatography technique, confirming the overhang domain protein expression.
4C is a purity measurement after purifying the Japanese encephalitis virus coat protein overhang domain using gel filtration chromatography technique.
Figure 4d shows the expression of the protruding domain protein after purifying the Japanese encephalitis virus coat protein protruding domain using gel filtration chromatography technique.
4E shows the expression of purified Japanese encephalitis virus coat protein overhang domain on SDS-PAGE.
Figure 5 shows the degree of immune antibody formation by the antigen after separating the final serum from four mice immunized with the Japanese encephalitis virus coat protein overhang domain antigen.
Fig. 6 shows the amount of antibody in the blood of each mouse administered with the Japanese encephalitis virus coat protein overhang domain antigens 2 (JEVx2) and 3 (JEVx3).
Figure 7 measures the specificity and sensitivity of insect cell-derived Japanese encephalitis virus coat protein overhang domain antigen (JEV-E) and E. coli expression system-derived antigen (LysRS-JEV-E).
Figure 8a is a measure of the degree of antibody production according to the inoculation method of the adjuvant and vaccine.
Figure 8b is a value quantified based on the mouse standard IgG value generated by the inoculation method of the immunostimulant and vaccine.

In one aspect, the present invention provides a homodimeric protein in which two Japanese encephalitis virus capsular protein protruding domains are combined.

As used herein, the term "Japanese encephalitis virus" is a virus belonging to the genus Flaviviridae flavivirus. The virus consists of a nucleocapsid composed of a complex of genomic RNA and a capsid protein and a coating protein surrounding the nucleocapsid. The coat protein is a factor that determines the serotype of the virus.

In addition, as used herein, the term "Japanese encephalitis virus coat protein" plays an important role in Japanese encephalitis virus infection and is a target of a vaccine or antibody that inhibits the activity of the virus. Therefore, the coat protein can be usefully used for the prevention or treatment of Japanese encephalitis.

The protruding domain may be a polypeptide having an amino acid sequence of SEQ ID NO: 3 or a fragment thereof, and the polypeptide may be encoded by the nucleotide sequence of SEQ ID NO: 2.

The protruding domain is subdomain 1 (FNCLGMGNRD, (SEQ ID NO: 4)), subdomain 2 (VLEGDSCLT, (SEQ ID NO: 5)), subdomain 3 (HASVTDIS, (SEQ ID NO: 6)), subdomain 4 (GWGNGCGL, ( SEQ ID NO: 7)), subdomain 5 (TTTSENHGN, (SEQ ID NO: 8)), subdomain 6 (AHATKQSVVA, (SEQ ID NO: 9)), subdomain 7 (QEGGLHQALAGAIVVEYS, (SEQ ID NO: 10)), and subdomain 8 ( DTGHGTV, (SEQ ID NO: 11)).

Specifically, for the amino acid sequence of SEQ ID NO: 3 representing the Japanese encephalitis virus capsular protein overhang domain, subdomain 1 may be located at 3 to 12; Subdomain 2 may be located at 26-34; Subdomain 3 may be located at 64-71; Subdomain 4 may be located at 102-109; Subdomain 5 may be located from 148 to 156; Subdomain 6 may be located from 247 to 256; Subdomain 7 may be located at 260-277; Subdomain 8 may be located at 318-324.

The subdomains 1 to 8 may be connected through a linker.

In this case, the linker (L1) connecting the subdomain 1 and the subdomain 2 may include 1 to 30, 2 to 25, 3 to 20, or 5 to 15 amino acids, preferably May be a peptide consisting of 13 amino acids. In one embodiment of the present invention, L1 is located at positions 13 to 25 of SEQ ID NO.

The linker L2 connecting the subdomain 2 and the subdomain 3 may include 1 to 50, 2 to 45, 3 to 40, 5 to 35, or 10 to 30 amino acids. And preferably a peptide consisting of 29 amino acids. In one embodiment of the present invention, L2 is located at positions 35 to 63 of SEQ ID NO: 3.

The linker L3 connecting the subdomain 3 and the subdomain 4 may include 1 to 50, 2 to 45, 3 to 40, 5 to 35, or 10 to 30 amino acids. And preferably a peptide consisting of 30 amino acids. In one embodiment of the present invention, L3 is located at 72 to 101 of SEQ ID NO: 3.

The linker L4 connecting the subdomain 4 and the subdomain 5 may include 1 to 60, 2 to 55, 3 to 50, 5 to 45, or 10 to 40 amino acids. And preferably a peptide consisting of 37 amino acids. In one embodiment of the present invention, L4 is located at 110 to 147 of SEQ ID NO.

The linker L5 connecting the subdomain 5 and the subdomain 6 may include 1 to 120, 20 to 110, 40 to 100, or 60 to 90 amino acids, preferably It may be a peptide consisting of 90 amino acids. In one embodiment of the present invention, L5 is located at 157 to 246 of SEQ ID NO.

The linker L6 connecting the subdomain 6 and the subdomain 7 may include 1 to 10, 2 to 8, or 3 to 5 amino acids, and preferably a peptide composed of 3 amino acids. Can be. In one embodiment of the present invention, L6 is located at Nos. 257 to 259 of SEQ ID NO.

The linker (L7) connecting the subdomain 7 and the subdomain 8 may include 1 to 60, 5 to 55, 10 to 50, or 20 to 40 amino acids, preferably It may be a peptide consisting of 40 amino acids. In one embodiment of the present invention, L7 is located at 278 to 317 of SEQ ID NO.

The amino acids constituting the linker are arginine (Arg), histidine (His), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), threonine (Thr), asparagine (Asn), glutamine ( Gln), tyrosine (Tyr), alanine (Ala), leucine (Leu), isoleucine (Ile), valine (Val), phenylalanine (Phe), methionine (Met), tryptophan (Trp), glycine (Gly), proline (Pro), and cysteine (Cys) may be any one or more selected from the group consisting of.

In another aspect, the present invention provides a homodimeric protein of a Japanese encephalitis virus coat protein protruding domain, to which two proteins comprising the Japanese encephalitis virus coat protein protruding domain are combined. The linkage may be at a site of SEQ ID NO: 4 to SEQ ID NO: 10 present in the protruding domain.

The present invention also provides a vaccine for preventing or treating diseases caused by Japanese encephalitis virus infection comprising the homodimer protein as an active ingredient.

The disease caused by the Japanese encephalitis virus infection may be selected from the group consisting of encephalitis, aseptic meningitis, febrile headache and combinations thereof.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier. The carrier is about 1% to about 99.99%, 5% to 90%, 10% to 85%, 20% to 60% by weight, preferably based on the total weight of the pharmaceutical composition of the present invention About 90% to about 99.99% by weight.

The present invention also provides a method for preventing or treating a disease caused by Japanese encephalitis virus infection comprising administering the vaccine to a subject.

The present invention also provides an antibody or fragment thereof that specifically binds to the homodimeric protein.

The term "antibody" is a protein produced by stimulation of an antigen in the immune system, which is a protein that specifically binds to a specific antigen and floats lymph and blood to generate an antigen-antibody reaction. Antigen-antibody responses have high specificity for each antigen. This is because when antibodies are made in B cells of lymphocytes, antibodies produced by specific antigens do not, in principle, react with other antigens. Such high specificity is used for the examination of immunity, allergy, various diseases and types and types of infections.

The term “antigen” refers to a protein expressed by a virus as a component capable of generating immune function among the components of the virus. In one embodiment of the present invention, the homodimeric protein of the Japanese encephalitis virus coat protein overhang domain acted as an antigen that induces immune antibody formation when administered to a subject.

The present invention also provides a kit for diagnosing a Japanese encephalitis virus infection comprising the homodimer protein, and a kit for diagnosing a Japanese encephalitis virus infection comprising an antibody specifically binding to the homodimer protein.

In one embodiment, the homodimeric protein of the Japanese encephalitis virus coat protein overhang domain can specifically bind to an antibody against the Japanese encephalitis virus, and thus can be applied to a Japanese encephalitis virus infection diagnostic kit.

The present invention also provides a Japanese encephalitis virus infection information providing method comprising the step of adding a sample isolated from the diagnosis target to the Japanese encephalitis virus infection diagnosis kit.

The term "Japanese encephalitis virus infection diagnosis kit" is a kit capable of rapidly diagnosing Japanese encephalitis virus infection and diseases thereby. Tests using these kits can know the test results within 10 to 15 minutes, and there is an advantage that anyone can easily diagnose a disease caused by Japanese encephalitis virus infection because no expert or equipment for diagnosis is needed.

In another aspect, the present invention provides an expression vector comprising a nucleotide encoding a Japanese encephalitis virus coat protein protruding domain monomer protein. In this case, the vector may be pAcGP67A-JEVE401.

In one embodiment of the present invention, the exodus domain of the Japanese encephalitis virus coat protein represented by SEQ ID NO: 3 was subcloned to the BamHI and ECoRI sites of the pAcGP67A vector and overexpressed using an E. coli strain. Then, the expression vector pAcGP67A-JEVE401 was purified using DNA midi-prep (Qiagen, Germany).

The present invention also provides a host cell transfected with the expression vector. The host cell may be a eukaryotic cell, and the eukaryotic cell may be an insect cell. In this case, the host cell may be further transfected by baculovirus.

In one embodiment of the present invention, the pAcGP67A-JEVE401 vector and baculovirus DNA were transfected into sf9 cells, which are insect cells, using a 10% profectin (AB vector), a transfection reagent.

The present invention also provides a method for producing a Japanese encephalitis virus coat protein protruding domain monomer protein comprising the step of obtaining a Japanese encephalitis virus coat protein protruding domain monomer protein from the transfected host cell.

It may comprise the step of purifying the obtained Japanese encephalitis virus coat protein protruding domain monomeric protein. In this case, the purification may be performed by affinity chromatography or gel filtration chromatography.

In one embodiment of the present invention, Japanese encephalitis virus coat protein protruding domain monomer protein was obtained by culturing insect cells transfected with the pAcGP67A-JEVE401 vector and baculo virus DNA.

In another aspect, the present invention, (1) inducing an immune response by injecting the homodimeric protein as an antigen into an experimental animal; And (2) provides a Japanese encephalitis virus antibody production method comprising the step of recovering the antibody from the experimental animal.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example  One. Flavi  Comparative Analysis of Encapsulating Protein Sequences of Virus and Japanese Encephalitis Virus

The protein sequencing comparison program (Clustal W & ESPript 3.0) was used to compare the coat protein amino acid sequences of Flavi virus and Japanese encephalitis virus. The results are shown in FIGS. 1 and 2.

As shown in Figures 1 and 2, the sequences of the Flavi virus and the Japanese encephalitis virus were compared to confirm the conserved sequence between the proteins, and designed a stable overhang domain based on the structure of the protein.

Example  2. Expression vector production and expression of Japanese encephalitis virus coat protein overhang domain

Nakayama strain (Yonsei University) was used as the Japanese encephalitis virus. The coat protein of the Nakayama strain was obtained by amplifying the gene directly in the virus, and the base sequence of the coat protein is shown in SEQ ID NO: 1.

The exudation domain of the Japanese encephalitis virus coat protein was subcloned into the BamHI and ECoRI sites of the pAcGP67A vector and Escherichia coli ) and the vector was amplified using the DH10β strain. Thereafter, the expression vector (pAcGP67A-JEVE401) was purified through DNA midi-prep (Qiagen, Germany). The pAcGP67A-JEVE401 vector and baculovirus DNA purified using a transfection reagent (10% profectin, AB vector) were transfected into sf9 cells, which are insect cells. After 50 hours, the virus supernatant containing pAcGP67A-JEVE401 was obtained. After passage five times in such a manner that the supernatant was reinfected at a ratio of 1:20, the expression of the protruding domain of the coat protein secreted into the culture supernatant was confirmed using Western blot. The results are shown in FIG.

Example  3. Mass expression of Japanese encephalitis virus coat protein overhang domain

For mass expression of the Japanese encephalitis virus coat protein overhang domain identified in Example 2, the culture solution of insect cell Hi5 cells incubated with 2.5% FBS was incubated to 1.5 X 10 ⁶ cells / ml to 2.0 X 10 ⁶ cell / ml. . The cultures were then virus infected at a ratio of 1:20 and incubated at 27 ° C. for 4 days. After incubation, the supernatant of the culture was centrifuged to obtain the overhang domain of the Japanese encephalitis virus coat protein.

Example  4. Purification of Japanese Encephalitis Virus Skim Protein Protruding Domain

For affinity chromatography purification using Ni-NTA of six histidine-labeled Japanese encephalitis virus coat protein overhang domains, a column equilibration buffer solution, pH 7.5, 20 mM Tris-HCl solution and 0.2 M NaCl solution for 3 days Dialysis procedure was performed. After dialysis, the supernatant containing the coat protein protruding domain was slowly bound to a nickel-nitrilotriacetic acid (Ni-NTA) agarose column. Then, the column was washed with the same buffer solution as above, and fractions were collected while eluting the protein by linear concentration method using a buffer solution containing 0.5 M imidazole. Thereafter, the eluted protein was confirmed through SDS-PAGE. The results are shown in FIGS. 4A and 4B.

The identified protein was concentrated to 5 ml through a concentrator (Vivaspin, MWCO 30KDa), and then superdex 200 (GE Healthcare, 16X600) gel filtration (Eel Filtration) equilibrated with a 2-fold concentration of phosphate buffered saline (PBS) buffer solution. Final purification using chromatography. The purified protein was quantified by Bradford assay (Bio-rad) method and concentrated to 1 mg / ml and compared with the standard protein 1 mg / ml bovine serum albumin on SDS-PAGE. The results are shown in Figs. 4c to 4d. 4E shows that the expression of the purified Japanese encephalitis virus coat protein overhang domain was confirmed on SDS. Thereafter, the purified protein was dispensed and stored at -80 ° C until the experiment.

As shown in Figures 4a to 4e, the Japanese encephalitis virus coating protein protruding domain prepared in Example 1 was purified at least 98% through the purification process by Ni-NTA affinity chromatography and gel filtration chromatography technique It became.

Example  5. Antibody Production Experiment

The antibody production experiment was carried out for the homodimeric protein antigen of the protruding domain of the Japanese encephalitis virus coat protein obtained through the purification process of Example 4. First, before injection of the homodimeric protein antigen into the mouse, pre-immune serum was collected and used as a negative control. Primary immunization was injected intraperitoneal (IP) with 200 μg of antigen, followed by primary boosting by mixing with incomplete freund's adjuvant (IFA) (sigma) two weeks later. Proceeded. After 1 week, primary blood was collected and the ELISA test was performed. Boosting and bleeding were performed at weekly intervals. One week after the third boosting, blood was collected from the heart of the mouse and the final serum was isolated to undergo a final ELISA test. In the ELISA test, the antigen was used coated with 100 ng / well. In addition, the serum collected and stored at each boosting time was diluted by a certain ratio using 0.05% tween PBS solution (hereinafter referred to as 0.2% skim milk) containing 0.2% skim milk, and diluted anti-mouse IgG at 1: 10,000. Antigen-specific antibodies were detected using -HRP (ABC5001).

Experimental Example  1. the bulging domain of a Japanese encephalitis virus coat protein Homodimer  Immune antibody formation experiment of protein antigen

1.1. Antibody Formation Experiment

In order to confirm the immune antibody formation of the homodimeric protein antigen of the protruding domain of the Japanese encephalitis virus coat protein, four homodimeric protein antigens (JEP401) of the protruding domain of the Japanese encephalitis virus coat protein purified in Example 4 were identified. Mice were immunized. After 4 weeks, the final serum was isolated from each mouse and subjected to the ELISA test. In the ELISA test, the antigen was used 100 ng per well, and the isolated immune serum was diluted in a ratio of 1: 100 to 1: 100,000 with 1 × PBS. In addition, anti-mouse IgG-HRP (ABC5001) was used for the secondary confirmation, and the color was detected and compared at the absorbance (Optical Density, OD) of 405 nm. This is shown in Table 1 and FIG.

Sample Dilution JEP401 Factor #One #2 # 3 #4 Pre-serum 1: 100 0.090 0.082 0.089 0.094

Test serum 1: 100 2.603 2.570 2.666 2.676 1: 1,000 2.475 2.371 2.231 2.384 1: 5,000 1.730 1.532 0.949 1.165 1: 10,000 1.256 1.047 0.605 0.903 1: 50,000 0.422 0.344 0.188 0.310 1: 100,000 0.289 0.250 0.149 0.249

As shown in Table 1 and Figure 5, the antibody concentration was increased in mice injected with homodimeric protein antigens of the protruding domains of the Japanese encephalitis virus coat protein. Through this, when injecting the homodimer protein antigen of the protruding domain of the Japanese encephalitis virus coat protein to the subject, it was found that the immune antibody against it is well produced and effective as a vaccine.

1.2. Of the Protruding Domain of the Japanese Encephalitis Virus Skim Protein Homodimer  Identification of antibody formation difference according to the number of immunization of protein antigen

In order to confirm the antibody formation difference according to the number of immunization of the homodimer protein antigen of the protruding domain of the Japanese encephalitis virus coat protein, 2 isomeric protein antigen of the protruding domain of the Japanese encephalitis virus Mice were immunized 2 and 3 times, respectively. Then, the antibody of Japanese encephalitis virus present in the serum of each mouse was measured using the ELISA technique. The results are shown in FIG.

As shown in FIG. 6, the concentration of antibody in the blood of the mouse immunized three times (JEVx3) was higher than that of the mouse immunized twice (JEVx2). This means that as the number of administration of the protruding domain antigen increases, its antibody formation increases.

1.3. Antibody-forming Clonal Cell Identification

Monoclonals of homodimeric protein antigens of the protruding domain of the Japanese encephalitis virus capsular protein were identified according to the method of Experimental Example 1.1, in order to identify the clonal cells forming antibodies of the homodimeric protein antigens of the protruding domain of the Japanese encephalitis virus coat protein. Antibodies were prepared for ELISA testing and isotyping screening. This is shown in Table 2 and Table 3.

Clone No. OD 1A8 2.011 1G1 1.836 1H4 2.015 4E12 1.825 5D8 1.754

1A8 1G1 1H4 4E12 5D8 IgG1 0.060 0.048 0.047 0.047 1.807 IgG2a 2.095 0.041 0.064 0.041 0.048 IgG2b 0.067 1.564 1.652 1.302 0.082 IgG3 0.046 0.041 0.041 0.041 0.041 IgA 0.041 0.040 0.041 0.039 0.040 IgM 0.040 0.041 0.040 0.040 0.041 Kappa 0.673 0.386 0.579 0.354 0.694 Lambda 0.044 0.043 0.044 0.041 0.043

Experimental Example  2. Confirmation of Homodimer Protein Antigen Specificity of the Protruding Domain of Insect Cell-derived Japanese Encephalitis Virus Coat Protein

In order to compare the specificity of the homodimeric protein antigen of the protruding domain of the insect cell-derived Japanese encephalitis virus coat protein and the antigen derived from E. coli expression system according to the present invention, a high concentration of serum (1:25) and antigen (200, 400, 800 ng / well) was used for the ELISA test. The results are shown in FIG.

As shown in FIG. 7, little antigen-antibody response was detected in the E. coli expression system-derived antigen (LysRS-JEV-E). On the other hand, the antigen-antibody response was high in homodimeric protein antigen (JEV-E) of the protruding domain of insect cell-derived Japanese encephalitis virus coat protein. This means that the insect cell-derived antigen of the present invention is significantly superior in sensitivity and specificity than the E. coli-derived antigen.

Experimental Example  3: of the overhang domain of the Japanese encephalitis virus coat protein Homodimer  Vaccine efficacy assessment

In order to confirm whether the homodimeric protein according to the present invention can be applied to the actual antibody in serum, Japanese encephalitis-positive mouse serum was prepared and secured. To compare the efficacy of purified proteins and the sensitivity and accuracy of experimental techniques for ELISA techniques, SOP ( Shirley HL et al., Journal of Virological Methods, 2001 ). A mouse-derived Japanese encephalitis-positive antiserum was used to compare the newly developed ELISA technique with the PRNT assay. The results are shown in Table 4, FIG. 8A, and FIG. 8B.

Immunizing virus Route of immunization Immunizing dose (TCID ₅₀ ) Neutralizing antibody NC - - -
SA ₁₄ -14-2 s.l. (+ cholera toxin) 2.5 x 10 ⁵ <160 i.m. 2.5 x 10 ⁵ <640
MVA (Vjh6) s.l. (+ cholera toxin) 1.0 x 10 ⁶ <160 i.m. 1.0 x 10 ⁶ <320

Table 4 shows the titer values of TCID ₅₀ and the neutralizing ability of the antibody using serum obtained by sublingual administration and intramuscular injection of the antigen and the adjuvant.

Figure 8a is a measure of the amount of anti-JEV serum of each sample through the ELISA using the serum obtained in the above experiment, the serum is serially diluted from 1: 100 to 1: 10,000 to measure the degree of the antibody in the serum It is.

FIG. 8B is a value obtained by quantifying the concentration of JEV antibody in the mouse using standard serum using the same serum sample as in FIG. 8A. It was found that MAV showed a better effect when comparing the two immune enhancers, SA-14-2 and MAV, and that the vaccine was administered intramuscularly (im) rather than sublingual (sl) vaccine. In the case of MAV, the sublingual vaccine also acts as an adjuvant to provide a good vaccine. This experiment can not only measure JEV antibody value in blood but also can be easily used as a diagnostic method in the development of vaccines and therapeutics.

As shown in Table 4, FIG. 8A, and FIG. 8B, significant values of anti-JEV IgG were measured in serum samples of mice administered JEV vaccine candidates, which values were neutralizing derived from TCID ₅₀ and PRNT analysis. It shows the same pattern as the antibody measurement value. This means that newly established ELISA-based antibodies have a level of confidence that Japanese encephalitis standard antibodies can be used as assays . In particular, the ELISA technique performed in this experiment is simpler than the conventional PRNT technique and is safe because it does not use live viruses. As a result, it is expected to be very useful in research and clinical practice.

<110> Korea Research Institute of Bioscience and Biotechnology          Industry-Academic Cooperation Foundation, Yonsei University <120> PROTEIN COMPRISING ECTODOMAIN OF JAPANESE ENCEPHALITIS VIRUS          ENVELOPE PROTEINS, HOMODIMER THEREOF, AND USE THEREOF <130> FPD / 201708-0036 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 1500 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence for Japanese encephalitis virus envelop          protein (strain: Nakayama) <400> 1 ttcaactgtc tgggaatggg caatcgtgac ttcatagaag gagccagtgg agccacttgg 60 gtggacttgg tgctagaagg agacagctgc ttgacaatta tggcaaacga caaaccaaca 120 ttggacgtcc gcatgatcaa catcgaagct gtccaacttg ctgaggtcag aagttactgc 180 tatcatgctt cagtcactga catttcgacg gtggctcggt gccccacgac tggagaagct 240 cacaacaaga agcgagctga tagtagctat gtgtgcaaac aaggcttcac tgatcgtggg 300 tggggcaacg gatgtggact tttcgggaag ggaagcattg acacatgtgc aaaattctcc 360 tgcaccagta aggcgattgg gagaacaatc cagccagaaa acatcaaata cgaagttggc 420 atttttgtgc atggaaccac cacttcggaa aaccatggga attattcagc gcaagttggg 480 gcgtcccagg cggcaaagtt tacagtaaca cccaatgctc cttcgataac cctcaaactt 540 ggtgactacg gagaagtcac actggactgt gagccaagga gtggactaaa cactgaagcg 600 ttttacgtca tgaccgtggg gtcaaagtca tttttggtcc acagggaatg gtttcatgat 660 ctcgctctcc cttggacgcc cccttcgagc acagcgtgga gaaacagaga actcctcatg 720 gaatttgaag aggcgcacgc cacaaaacag tccgttgttg ctcttgggtc acaggaagga 780 ggcctccatc aggcgttggc aggagccatc gtggtggagt actcaaactc agtgaagtta 840 acatcaggcc acctaaaatg caggctgaga atggacaaac tggctctgaa aggcacaacc 900 tatggcatgt gcacagaaaa attctcgttc gcgaaaaatc cggcggacac tggtcacgga 960 acagttgtca ttgaactttc ctactctggg agtgatggcc cttgcaaaat tccgattgtc 1020 tccgttgcga gcctcaatga catgaccccc gtcgggcggc tggtgacagt gaaccccttc 1080 gtcgcgactt ccagcgccaa ctcaaaggtg ctagtcgaga tggaaccccc cttcggagac 1140 tcctacatcg tagttggaag gggagacaag cagattaacc accattggca caaggctgga 1200 agcacgctgg gcaaagcctt ttcaacgact ttgaagggag ctcaaagact ggtagcgttg 1260 ggcgacacag cctgggactt tggctctatt ggaggggttt tcaactccat agggaaagcc 1320 gttcaccaag tgtttggtgg tgccttcaga acactcttcg ggggaatgtc ttggatcaca 1380 caagggctaa tgggggccct actactctgg atgggcgtca acgcacgaga ccgatcaatt 1440 gctttggcct tcttagccac aggaggtgtg ctcgtgttct tagcgaccaa tgtgcatgct 1500                                                                         1500 <210> 2 <211> 1203 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence for ectodomain of Japanese encephalitis          virus envelop protein (strain: Nakayama) <400> 2 ttcaactgtc tgggaatggg caatcgtgac ttcatagaag gagccagtgg agccacttgg 60 gtggacttgg tgctagaagg agacagctgc ttgacaatta tggcaaacga caaaccaaca 120 ttggacgtcc gcatgatcaa catcgaagct gtccaacttg ctgaggtcag aagttactgc 180 tatcatgctt cagtcactga catttcgacg gtggctcggt gccccacgac tggagaagct 240 cacaacaaga agcgagctga tagtagctat gtgtgcaaac aaggcttcac tgatcgtggg 300 tggggcaacg gatgtggact tttcgggaag ggaagcattg acacatgtgc aaaattctcc 360 tgcaccagta aggcgattgg gagaacaatc cagccagaaa acatcaaata cgaagttggc 420 atttttgtgc atggaaccac cacttcggaa aaccatggga attattcagc gcaagttggg 480 gcgtcccagg cggcaaagtt tacagtaaca cccaatgctc cttcgataac cctcaaactt 540 ggtgactacg gagaagtcac actggactgt gagccaagga gtggactaaa cactgaagcg 600 ttttacgtca tgaccgtggg gtcaaagtca tttttggtcc acagggaatg gtttcatgat 660 ctcgctctcc cttggacgcc cccttcgagc acagcgtgga gaaacagaga actcctcatg 720 gaatttgaag aggcgcacgc cacaaaacag tccgttgttg ctcttgggtc acaggaagga 780 ggcctccatc aggcgttggc aggagccatc gtggtggagt actcaaactc agtgaagtta 840 acatcaggcc acctaaaatg caggctgaga atggacaaac tggctctgaa aggcacaacc 900 tatggcatgt gcacagaaaa attctcgttc gcgaaaaatc cggcggacac tggtcacgga 960 acagttgtca ttgaactttc ctactctggg agtgatggcc cttgcaaaat tccgattgtc 1020 tccgttgcga gcctcaatga catgaccccc gtcgggcggc tggtgacagt gaaccccttc 1080 gtcgcgactt ccagcgccaa ctcaaaggtg ctagtcgaga tggaaccccc cttcggagac 1140 tcctacatcg tagttggaag gggagacaag cagattaacc accattggca caaggctgga 1200 agc 1203 <210> 3 <211> 411 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence for ectodomain of Japanese encephalitis virus          envelop protein (strain: Nakayama) <400> 3 Gly Ser Phe Asn Cys Leu Gly Met Gly Asn Arg Asp Phe Ile Glu Gly   1 5 10 15 Ala Ser Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly Asp Ser Cys              20 25 30 Leu Thr Ile Met Ala Asn Asp Lys Pro Thr Leu Asp Val Arg Met Ile          35 40 45 Asn Ile Glu Ala Val Gln Leu Ala Glu Val Arg Ser Tyr Cys Tyr His      50 55 60 Ala Ser Val Thr Asp Ile Ser Thr Val Ala Arg Cys Pro Thr Thr Gly  65 70 75 80 Glu Ala His Asn Lys Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys Gln                  85 90 95 Gly Phe Thr Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys             100 105 110 Gly Ser Ile Asp Thr Cys Ala Lys Phe Ser Cys Thr Ser Lys Ala Ile         115 120 125 Gly Arg Thr Ile Gln Pro Glu Asn Ile Lys Tyr Glu Val Gly Ile Phe     130 135 140 Val His Gly Thr Thr Thr Ser Ser Glu Asn His Gly Asn Tyr Ser Ala Gln 145 150 155 160 Val Gly Ala Ser Gln Ala Ala Lys Phe Thr Val Thr Pro Asn Ala Pro                 165 170 175 Ser Ile Thr Leu Lys Leu Gly Asp Tyr Gly Glu Val Thr Leu Asp Cys             180 185 190 Glu Pro Arg Ser Gly Leu Asn Thr Glu Ala Phe Tyr Val Met Thr Val         195 200 205 Gly Ser Lys Ser Phe Leu Val His Arg Glu Trp Phe His Asp Leu Ala     210 215 220 Leu Pro Trp Thr Pro Pro Ser Ser Thr Ala Trp Arg Asn Arg Glu Leu 225 230 235 240 Leu Met Glu Phe Glu Glu Ala His Ala Thr Lys Gln Ser Val Val Ala                 245 250 255 Leu Gly Ser Gln Glu Gly Gly Leu His Gln Ala Leu Ala Gly Ala Ile             260 265 270 Val Val Glu Tyr Ser Asn Ser Val Lys Leu Thr Ser Gly His Leu Lys         275 280 285 Cys Arg Leu Arg Met Asp Lys Leu Ala Leu Lys Gly Thr Thr Tyr Gly     290 295 300 Met Cys Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Thr Gly 305 310 315 320 His Gly Thr Val Val Ile Glu Leu Ser Tyr Ser Gly Ser Asp Gly Pro                 325 330 335 Cys Lys Ile Pro Ile Val Ser Val Ala Ser Leu Asn Asp Met Thr Pro             340 345 350 Val Gly Arg Leu Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser Ala         355 360 365 Asn Ser Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser Tyr     370 375 380 Ile Val Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His Lys 385 390 395 400 Ala Gly Ser Leu Glu His His His His His His                 405 410 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop          protein <400> 4 Phe Asn Cys Leu Gly Met Gly Asn Arg Asp   1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop          protein <400> 5 Val Leu Glu Gly Asp Ser Cys Leu Thr   1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop          protein <400> 6 His Ala Ser Val Thr Asp Ile Ser   1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop          protein <400> 7 Gly Trp Gly Asn Gly Cys Gly Leu   1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop          protein <400> 8 Thr Thr Thr Ser Glu Asn His Gly Asn   1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop          protein <400> 9 Ala His Ala Thr Lys Gln Ser Val Val Ala   1 5 10 <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop          protein <400> 10 Gln Glu Gly Gly Leu His Gln Ala Leu Ala Gly Ala Ile Val Val Glu   1 5 10 15 Tyr ser         <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Binding site on ectodomain of Japanese encephalitis virus envelop <400> 11 Asp Thr Gly His Gly Thr Val   1 5

Claims (16)

Japanese encephalitis virus (JEV) encapsulated protein produced in insect cells, a protein consisting of a protruding domain, wherein the protruding domain is a polypeptide consisting of the amino acid sequence of 3 to 403 position of SEQ ID NO: 3, protein. The method of claim 1,
The protruding domain is FNCLGMGNRD (SEQ ID NO: 4), VLEGDSCLT (SEQ ID NO: 5), HASVTDIS (SEQ ID NO: 6), GWGNGCGL (SEQ ID NO: 7), TTTSENHGN (SEQ ID NO: 8), AHATKQSVVA (SEQ ID NO: 9), QEGGLHQALAGAIVVEYS (SEQ ID NO: 10), and a amino acid sequence of DTGHGTV (SEQ ID NO: 11). delete The method of claim 1,
The polypeptide consisting of the amino acid sequence of the position 3 to 403 of SEQ ID NO: 3, characterized in that encoded by the nucleotide sequence of SEQ ID NO: 2. A vaccine for preventing or treating diseases caused by Japanese encephalitis virus infection comprising the protein of claim 1 as an active ingredient. The method of claim 5,
Wherein said disease is selected from the group consisting of encephalitis, aseptic meningitis, febrile headache and combinations thereof. An antibody or fragment thereof that specifically binds to the protein of claim 1. Japanese encephalitis virus infection diagnostic kit comprising the protein of claim 1. Japanese encephalitis virus infection diagnostic kit comprising the antibody of claim 7. delete delete delete delete delete delete (1) inducing an immune response by injecting the protein of claim 1 into an experimental animal as an antigen; And
(2) Japanese encephalitis virus antibody production method comprising the step of recovering the antibody from the experimental animal.

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